Dehydrogenases, classified under E.C. 1.1, are enzymes that reversibly catalyse the oxidation or reduction of certain compounds such as for example alcohols and ketones. Some of the known dehydrogenases are cofactor-dependent, in other words they require further molecules to balance the electron transfer processes. NADH and NADPH for example are known to be such cofactors.
NADH is formed in the oxidation of, for example, alcohols to ketones according to the following reaction scheme. In order to avoid having to add stoichiometric amounts of expensive NAD+, the formation of NAD+ can be initiated by a second enzyme system (Enzyme Catalysis in Organic Synthesis, Ed.: K. Drauz, H. Waldmann, VCH, 1st Edition, p. 721; reaction scheme 1).

Systems known previously described using lactate dehydrogenase and pyruvate with the formation of lactate, or glutamate dehydrogenase with ketoglutarate and ammonium with the formation of glutamate for regenerating NAD+. The disadvantage of these systems is that auxiliary substrates, such as pyruvate or ketoglutarate, have to be used and products, such as lactate or glutamate, are formed, which for preparative utilization have to be removed from the product. In addition, equilibrium reactions go to completion only with great technical difficulty.
Alternatively, a second enzyme system may be an NADH oxidase that accepts atmospheric oxygen as oxidised cosubstrate and at the same time generates water or hydrogen peroxide with the formation of NAD+. In this respect, NADH oxidases are advantageous in that the reaction catalyzed by them is irreversible, O2 is used as regeneration substrate, and H2O or H2O2 are formed as product. Enzymes of both groups (H2O-forming and H2O2-forming enzymes) are, in principle, known in the biochemical literature (for example, for H2O-forming enzymes see Lopez de Felipe, F. et al., J. Bacteriol. Vol. 180 (1998), 3804-08; for H2O2-forming enzymes see Nishiyama, Y. et al. J. Bacteriol. Vol. 183 (2001), 2431-2438 and the literature cited therein). H2O2-forming enzymes are less suitable as a regeneration enzyme since peroxide is known to be harmful to enzymes and should be decomposed as far as possible in situ, which requires further, disadvantageous but not impossible process steps.
The NADH oxidases from Enterococcus (and Streptococcus) faecalis (Schmidt, H. L. et al., Eur. J. Biochem. 156 (1986), 149-55), Leuconostoc mesenteroides (Koike, K. J. et al., Biochem. (Tokyo) 97 (1985), 1279-88), Streptococcus mutans (Higuchi, M. et al., Biosci. Biotechnol. Biochem. 58 (1994), 1603-07), Mycoplasma capricolum (Klomkes, M., Altdorf, R., Ohlenbusch, H. D., Biol. Chem. Hoppe Seyler 366 (1985), 963-9), Sulfolobus solfataricus (Arcari, P. et al., J. Biol. Chem. 275 (2000), 895-900), Thermus thermophilus (Erdmann, H. et al., J. Mol. Biol. 230 (1993), 1086-8) and from Thermus aquaticus (Cocco, D. et al., Eur. J. Biochem. 174 (1988), 267-71) have been biochemically characterised.
However, there remains a need in the art for NADH oxidase with high activity, stability, etc. that when coupled with a dehyrogenase can be used for NAD+ regeneration.